Potential competitive and irreversible inhibitors will be utilized in examining the active site of the enzyme system involved in the biosynthesis of estrogens. First, biochemical studies on irreversible inhibitors of aromatase will be performed. The compounds to be studied are affinity analogs of the potent competitive inhibitor 7alpha-(4'-amino)phenylthioandrost-4-ene-3,17-dione (Brueggemeier et al., 1978). The aromatase preparation isolated in the microsomal fraction of human term placenta and a solubilized and partially purified enzyme preparation will be used to evaluate these compounds. Initial velocity, inactivation, and protection studies will be performed and K inact determined. Radiolabeled irreversible inhibitors will be synthesized and used to examine the irreversible nature of the inhibitor-enzyme interaction by gel chromatography, gel electrophoresis, and peptide mapping. Also, new 7-substituted androstenedione derivatives which do not contain the 7alpha-thioether linkage will be prepared and Ki's determined. These proposed studies will aid in examining the active site of aromatase and determining if more than one active site exists. The protection studies will also provide information on whether more than one aromatase exists for different substrates. Finally, aromatase inhibitors have potential therapeutic uses in the control of reproduction and in the treatment of estrogen-dependent cancers.